Antisense (i.e. sequences complementary to the "sense" strand, usually the messenger RNA) and otherwise interfering (e.g. "decoy") oligonucleotides, oligoribonucleotides, and modified oligonucleotides (collectively referred to herein as ASOs) are gaining overwhelming popularity for interference in various steps leading from DNA transcription to mRNA translation. This regulatory interference can be harnessed for therapeutic effects against many diseases and viral infections. Production of viral proteins can be inhibited by oligonucleotides that are complimentary to portions of mRNA for that protein (i.e. antisense). Other regulatory mechanisms of viruses, such as HIV, are also open to interference with the use of complimentary (i.e. antisense), or decoy (i.e. sense) oligonucleotides. Oligonucleotides for therapy have several advantages. These advantages include the extremely high specificity and the ease of design afforded by Watson-Crick base pairing. The high association constants imply a strong duplex formation and thus effectiveness at low concentrations.
An ideal therapeutic agent should be (a) specific for the target, (b) selective for the target (i.e. minimum nonspecific effects), (c) non-toxic to the host, with a large therapeutic window, (d) effective at low concentrations, and (e) effective at eliminating the pathogen, rather than merely reducing the pathogen level.
For ASO therapy, several criteria are important. (i) The ASOs must be stable to degradation in vivo. (ii) The ASOs must be efficiently taken up by the cells. (iii) The ASOs must be retained by the cells. (iv) The ASOs must effectively interact with the target. (v) The ASOs should not non-specifically interact with other cellular factors. (vi) The cost of production should be low. (vii) The ASOs should have low toxicity, immunogenicity, mutagenicity, and should have a large therapeutic window. (viii) In addition, an ASO that allows RNaseH activity upon binding its target has a significant advantage as antisense to MnRNA, since the mRNA in the mRNA-ASO hybrid may be hydrolyzed by RNaseH, thereby destroying the mRNA, and releasing the ASO, thus creating a catalytic cycle. This is especially important in the case of many viral infections, such as HIV, since the reverse transcriptase; which is localized primarily in the cytoplasm itself, has RNaseH activity. Cellular RNaseH has been thought to be localized primarily in the nucleus, and to some extent in the cytoplasm as well. Natural phosphodiester ASOs are subject to nuclease activity and therefore possess a short half-life.
Natural oligonucleotides and their several modified analogs are widely used as tools for regulating specific gene expression (Wickstrom, E. L., Bacon, T. A., Gonzalez, A., Freeman, D. L., Lyman, G. H., Wickstrom, E., Proc, Natl. Acad. Sci., USA, 85, 1028-1032 (1988). They have been used for blocking, splicing and translation of mRNA (Blake, K. R., Murakami, A., Miller, P. S., Biochemistry, 24, 6132-6138 (1985); Haeuptle, M. T., Frank, R., Dobberstein, B., Nucl. Acids Res., 14, 1427-1448 (1986); Gupta, K. C., J. Biol. Chem, 262, 7492-7496 (1987); Maher, L. J., Dolnick, B. J., Nucl. Acids Res., 16, 3341-3358 (1988)). Originally the work of Zamecnik showed that a large excess of a natural antisense oligonucleotide can block the replication of Rous Sarcoma virus in chick fibroblast cells (Zameknik, P. C., and Stephenson, M. L., Proc. Natl. Acad. Sci., USA, 75, 285-288 (1978). This work was followed by other workers (Smith, C. C., Aurelain, L., Reddy, M. P., Miller, P. S., and Ts'O, P. O. P., Proc. Natl. Acad. Sci., USA, 83, 2787-2791 (1986); Zerial, A., Thoung, N. T., Helen, C., Nucl. Acids Res., 15, 9909-9919 (1987). Natural phosphodiester deoxy and ribonucleotides suffer from the disadvantage of rapid degradation by intracellular enzymes. Antisense oligos fall into two basic categories with respect to charge, charged, and uncharged molecules. There are distinct advantages for both types of molecules depending upon the cell types desired and specific use.
RNaseH has been used for cleaving an RNA strand of RNA-DNA hybrid at several positions when preparing small fragments from the large RNA molecules (Stein, H., and Hausen, P., Science, 166, 393-395 (1969).; Hausen, P., and Stein, H., Eur. J. Biochem. 14, 278-283, (1970).; Henry, C. M., Ferdinand, F. J., and Knippers, R., Biochem. Biophys. Res. Conl., 50, 603-611 (1973).; Donis-Keller, H., Nucl. Acids Res., 7, 179-192 (1979).; Ruskin, B., Krainer, A. R., Maniatis, T., and Green, M. R., Cell, 38, 317-331 (1984).; Schmelzer, C. and Schweyen, R. J., Cell, 46, 557-565 (1986).)
Synthetic short DNA oligomers (tetramers to hexamers) complimentary to a specific site in the RNA molecule, have been employed from the method of Donis-Keller, ibid. Using complimentary chimeric (hybrid) oligonucleotides containing deoxyoligonucleotides and 2'-O-methylribonucleotides, a site directed cleavage of enzymatically synthesized 90-mer RNA at a single site was achieved with E. coli RNaseH (Shibahara, S., Muhai, S., Nishihara, T., Inoue, H., Ohtsuka, E., Morisawa, H., Nucl. Acids Res., 15, 4403-4415 (1987)). At the same time it should be noted that 2'-O-methylribonucleotides containing RNA-RNA duplex is not a substrate for RNaseH. 2'-O-methylriboligonucleotides have been used in the purification of RNA-protein complexes from crude nuclear extracts (Blencowe, B. J., Sproat, B. S., Barbino, S., and Lamond, A. I., Cell, 59, 531-539 (1989). and Barbin, S., Sproat, B. S., Ryder, U., Blencowe, B. J., and Lamond, A. I., EMBO Journal, 8, 4171-4178 (1989)). The presence of 2'-O-methylribonucleotides in an RNA confers protection against nuclease digestion and are completely resistant to degradation by either RNA or DNA specific nucleases (Sproat, B. S., Lamond, A. I., Beijer, B., Neuner, P., and Ryder, U., Nucl. Acids Res., 17, 9, 3373 (1989)) and against alkaline hydrolysis. Similarly, it has also been demonstrated that a 2'-O-methyloligoribonucleotide-RNA duplex is much more thermally stable than the corresponding oligodeoxynucleotide-RNA duplex (Inoue, H., Imura, A., Iwai, S., Miura, M., Ohtsuka, E., Nucl. Acids Res., 15, 6131-6148 (1989)). Furthermore, as pointed out above, the former duplex is not a substrate for RNaseH (Inoue, H., Hayase, Y., Iwai, S., Ohtsuka, E., FEBS Letters, 215, 327-330 (1987)).
Phosphorothioate oligonucleotides are another modification of natural oligodeoxy and oligoribonucleotides which were shown to possess antiviral activity (Matsukura, M., Shinozuka, K., Zon, G., Mitsuya, H.; Reitz, M., Cohen, J. S., Broder, S., Proc. Natl. Acad. Sci., USA, 84, 7706-7710 (1987)); (Matsukura, M., Shinozuka, K., Zon, G., Mitsuya, H., Wong-Staal, F., and Cohen, J. S., Clinical Res., 36, 463A (1988)). It has been demonstrated that phosphorothioate DNA can function by activating endogenous RNaseH that will cleave the targeted RNA (Agarwal, S., Maynard, S. H., Zamecnik, P. C., Paderson, T., Proc. Natl. Acad. Sci., USA, 87, 1401-1405 (1990); Woolf, T. M., Jennings, C. G., Rebagliati, M., and Melton, D. A., Nucl. Acids Res., 18, 1763-1769 (1990)). With the notable exception of phosphorothioate DNA, the vast majority of nuclease resistant modified DNA backbone are not recognized by RNaseH. While phosphorothioate DNA has the advantage of activating RNaseH, it has the disadvantage of non-specific effects and reduced affinity for RNA. (Stein, C. A., Matsukara, M., Subasinghe,. C., Broder, S., Cohen, J. S., Aids Res. Hum. Retroviruses, 5, 639-646 (1989); Woolf, T. M., et al, ibid). Chimeric (also called gapmer) antisense oligos that have a short stretch of 6-9 nucleotides have been used to obtain RNaseH mediated cleavage of the target RNA, while reducing the number of phosphorothioate linkages (Dagle, J. M., Walder, J. A., Weeks, D. L., Nucl. Acids Res., 18, 4751-4757 (1990); Agarwal, S., Maynard, S. H., Zamecnik, P. C., Paderson, T., Proc. Natl. Acad. Sci., USA, 87, 1401-1405 (1990)). The chimeric oligonucleotides (gapmers or hybrids) have been designed in the past to have a central region that forms a substrate for RNaseH that is flanked by hybridizing "arms", comprised of modified nucleotides that do not form substrate for RNaseH. In these chimeras (gapmers or hybrids), the substrate for RNaseH that forms the "gap" can be on the 5'- or 3'-side of the oligomer.
The "arm" which does not form a substrate for RNaseH has the following relevant properties. They hybridize to the target providing the necessary duplex affinity to achieve antisense inhibition. They reduce the number of phosphorothioate DNA linkages in the oligomer, thus reducing non-specific effects. Lastly, they limit the region that forms a substrate for RNaseH, thus adding to the target specificity of the oligomers. Several chemistries have been used to make the regions of a chimeric oligomer that are not a substrate for RNaseH. The first chimeric oligomers used methyl phosphonate or phosphoramidate linkages in the arms ((Dagle, J. M., Walder, J. A., Weeks, D. L., Nucl. Acids Res., 18, 4751-4757 (1990); Agarwal, S., Maynard, S. H., Zamecnik, P. C., Paderson, T., Proc. Natl. Acad. Sci., USA, 87, 1401-1405 (1990)).
While these compounds functioned well in buffer systems and Xenopus oocytes, the "arm" decreased the hybrid affinity. This decrease in affinity dramatically reduced the activity of oligomers in mammalian culture.
Methyl phosphonate modified oligodeoxy and ribonucleotides have been employed as antisense in the past. The replacement of an oxygen by a methyl group in methyl phosphonate oligodeoxynucleotides disturbs the conformation around the P-O3 and P-O5 linkages, due to stereoelectronic factors. Therefore, longer (&gt;8 nucleotides) methyl phosphonate systems do not readily adopt a helical geometry, resulting in poor hybridization with natural DNA. Indeed, it has been found that methyl phosphonate. 21-niers have a low antisense activity, compared to shorter oligos (Marcus-Sekura, C. J., Anal. Biochem., 172, 289-295 (1988). Many oligos containing methyl phosphonate backbone oligodeoxyribonucleoside methylphosphonates of defined sequence were prepared and extensively characterized (Miller, P. S., Reddy, P. M., Murakami, A., Blake, K. R., Lin, S. B., Agris, C., Biochemistry, 25, 5092 (1986)). They were shown to be resistant to several exonucleases (Agarwal, S., Goodchild, J., Tet. Lett., 28, 3539 (1987)). The methyl phosphonates were shown to hybridize with mRNA in Agarose gel (Murakami, A., Blake, K. R., Miller, P. S., Biochemistry, 24, 4041 (1985)).
Methylphosphonate oligodeoxynucleotides (MP), complementary to specific regions of the bcr-abl mRNA of Philadelphia chromosome, present in chronic myelogenous leukemia (CML) condition, were incorporated in liposomes. The liposomal MP (L-MP) caused inhibition of the growth of CML cells (Tari, A. M., Tucker, S. D. T., Deisseroth, A., Berestein, G. L., Blood, 84, 601-607 (1994).
Phosphoramidates, such as N-methoxyphosphoramidate oligonucleotides, have been synthesized. However, they form less stable duplexes with complimentary sequences, when compared to "all diester" oligomers. The analog, however, was found to be highly resistant to nucleases (Peyrottes, S., Vasseur, J. J., Imbach, J. L., Rayner, B., Nucleosides and Nucleotides, 13, 2135-3149 (1994)). Other reports have shown high stability to enzymatic cleavage but lower binding affinity for the DNA and RNA targets (Froehler, B. C., Tet. Lett., 27, 5575 (1986); Froehler, B. C., Matteucci, M. D., Nucl. Acids. Res., 16, 4831 (1988); Jager, A., Levy, M. J., Hecht, S. M., Biochemistry, 27, 7237 (1988); Letsinger, R. L., Singman, C. N., Histand, G., Salunkhe, M., J. Am. Chem. Soc., 110, 4470 (1988); Agarwal, S., Goodchild, J., Civeria, M. P., Thornton, A. H., Sarin, P. S., Zamecnik, P. C., Proc. Natl. Acad. Sci., USA, 85, 7079, (1988)). These modifications are not able to direct RNaseH cleavage.
A number of studies have been reported for the synthesis of phosphate alkylated (methyl and ethyl phosphotriester) oligonucleotides and their physico-chemical and biochemical evaluation. Dinucleotides with methyl and ethyl triesters were shown to possess greater affinity towards polynucleotides possessing complementary sequences (Miller, P. S., Fang, K. N., Kondo, N. S., Ts'O, P. O. P., J. Am Chem. Soc., 93, 6657, (1971)). However, a few years ago, another group reported lack of, or poor binding affinity of, heptethyl ester of oligothymidine with complementary polynucleotides (Pless, R. C., and Ts'O, P. O. P., Biochemistry, 16, 1239-1250 (1977)). Phosphate methylated oligonucleotides were synthesized and found to possess resistance towards endonuclease digestion (Gallo, K. L., Shao, K. L., Phillips, L. R., Regan, J. B., Kozioikiewicz, Uznanski, B., Stec, W., Zon, G., Nucl. Acids Res., 18, 7405 (1986)). A phosphate methylated 18-mer oligonucleotide was shown to have high Tm value in duplexes with natural DNA and blocked the DNA replication process at room temperature (Moody, H. M., van Genderen, M. H. P., Koole, L. H., Kocken, H. J. M., Meijer, E. M., Buck, H. M., Nucl. Acids Res., 17, 4769-4782 (1989)). These authors postulated that phosphate ethylated oligonucleotides would have poor antisense properties. Phosphate methylated dimers of DNA bases were synthesized using transient protecting group of FMOC for the exocyclic amino groups (Koole, L. H., Moody, H. M., Broeders, N. L. H. L., Quaedflieg, P. L. L. M., Kuijpers, W. H. L., van Genderen, M. H. P., Coenen, A. J. J. M., vanderWal, S., Buck, H. M., J. Org. Chem, 54, 1657-1664 (1989)).
Synthesis and physico-chemical properties of partial methylated oligodeoxyribonucleotides were determined. Only the thymidine and cytidine oligomers with methyl phosphotriester could be prepared satisfactorily due to difficulty in maintaining methyl triester intact. Furthermore, the methyl group was found to have a destabilizing effect on the hybridization properties of the modified oligomers with its complementary sequence by comparison with the unmodified parent oligodeoxynucleotide (Vinogradeov, S., Asseline, U., Thoung, N. T., Tet. Lett., 34, 5899-5902 (1993)).
Few other reports have postulated that phosphate methylated oligonucleotides are preferable as antisense DNA based on stronger hybridization than methyl phosphonate or phosphate ethylated systems (van Genderen, M. H. P., Koole, L. H., Merck, K. B., Meijer, E. M., Sluyterman, L. A. A. E., Buck, H. M., Proc. Kon. Ned Akad. van Wetensch., B90,155-159 (1987); van Genderen, M. H. P., Koole, L. H., Buck, H. M., Recl. Trav. Chim. Pays Bas, 108, 28-35 (1989)). The later authors carried out certain protein binding studies with p-methoxy and p-ethoxy oligonucleotides and postulated on the hypothesis of protein induced DNA duplex destabilization by such oligonucleotides.
Phosphate ethylated systems were shown by the same authors to display comparatively poor hybridizing properties, and postulated to have poor antisense properties (Moody, H. M., van Genderen, M. H. P., Koole, L. H., Kocken, H. J. M., Meijer, E. M., Buck, H. M., Nucl. Acids Res., 17, 4769-4782 (1989)). p-isopropoxyphosphoramidites containing p-O-methyl have been synthesized from several nucleosides (Stec, W. J., Zon, G., Gallo, K. A., Byrd, R. A., Tet. Lett., 26, 2191-2194 (1985)), and a few short oligonucleotides were synthesized, and hybridization studies were carried out. A few short oligonucleotides containing p-O-methyl were synthesized using 2-(acetoxymethyl) benzoyl (AMB) as a nucleoside base protecting group. (Kuijpers, W. H. A., Huskens, J., van Boeckel, C. A. A., Tet. Lett., 31, 6729-6732 (1990)). However, no biological activity or related properties were reported. These authors have further reported the application of AMB protecting group to synthesize methyl phosphonate containing oligodeoxynucleotides (Kuijpers, W. H. A., Yeheskiely, E. K., van Boom, L. H., van Boeckel, C. A. A., Nucl. Acid Res., 21, 3493-3500 (1993)).